Nosocomial infections by multiple antibiotic resistant bacteria are especially difficult to cure, pose a significant health risk and place an enormous burden on the economy. A leading cause of such nosocomial infections is the Gram-positive bacterium Enterococcus faecalis, which ranks high among the most commonly encountered pathogens infecting the bloodstream, surgical sites and urinary tract. In spite of E. faecalis being a leading cause of nosocomial infections, little is known about the bacterial factors involved in promoting persistence of enterococci in the nosocomial environment or at infection sites. We recently identified the first pathogenicity island in Enterococcus faecalis. Among the virulence traits that defined this element as a pathogenicity island is a gene encoding the surface protein Esp. Esp is enriched among infection-derived enterococcal isolates and has a unique architecture with multiple tandem repeat motifs, a feature characteristic of many bacterial surface protein adhesins involved in binding to host ligands. In previous studies we have determined that due to the variation in the number of tandem repeat units within the structural esp gene, E. faecalis cells may express Esp of varying size and structure at the cell surface. Preliminary studies indicate that alterations in the structure of Esp may influence its role in biofilm formation by enterococci on abiotic surfaces, and in colonization of the urinary bladder during infection. The specific aims of this proposal have been formulated to explore these dual functionalities of Esp. We propose to construct mutants lacking specific modular domains of Esp in an isogenic background, and assess the role of each domain in formation of enterococcal biofilms to determine which elements of Esp structure are essential for biofilm formation by E. faecalis. We also propose to construct and screen a library of transposon-insertion mutants to identify and characterize additional gene products that may play a role in the biofilm forming property of E. faecalis. Mutants identified in these studies will be evaluated to ascertain if mutations that affect biofilm formation also affect interaction with the host urothelium. Finally, urothelial cell receptors that may be bound by Esp will be identified and the role of the various Esp modular domains in colonizing urothelial surfaces will be determined. The long term goals of the proposed research are to be able to better identify and control infections by pathogenic enterococci and to identify new enterococcal targets for therapeutic intervention.